Host protein CD63 enhances viral RNA replication by interacting with human astrovirus nonstructural protein nsP1a/4

wei zhao; Nian liu; Xiao Li Tao; Chun hong Zheng; Xiang yu Li; Man Yu; Yong gang Li

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/331835 doi: bioRxiv preprint the interactionsbetween HAstV nsP1a and host proteins using a protein-protein 91 overlay assay. We amplified thensP1a gene by PCR and inserted the resulting 92 PCR product (2781bp) by recombination cloning into the yeast two-hybrid 93 (Y2H) bait vector pGBKT7 as a C-terminal fusion with a Gal4 DNA-binding 94 domain (BD). We confirmed the correct constructs by enzyme digestion and 95 DNA sequence analysis (Fig 1A) . When introduced alone into yeast Y2HGold 96 cells, the Gal4 (BD)-nsP1a bait construct wasnot toxic to the yeast and did not 97 auto-activate the Y2H reporter gene, indicating that the construct was suitable 98 for use in a Y2H screen (Fig 1B) . 99 Using a Y2H assay with Gal4 (BD)-nsP1a as bait and a human Caco-2 cDNA 100 prey library, we screened approximately 2.5×10 5 clones and identified 14 101 positive bait-prey interactions. Sequence and bioinformatics analysis of the 14 102 positive prey plasmids,listed by their initial positive-interaction identification 103 number,indicated that they represented 14 different human cDNAs(data not 104 shown). We next measured the strength of interaction between thensP1a bait and 105 each of the 14 human prey proteins in yeast using a qualitative growth assay and 106 quantitative β-galactosidase activity assays. We performed bioinformatic 107 analysis of the human proteins confirmed by the qualitative growth assay to gain 108 functional insights into the potential interactions between those proteins and 109 nsP1a (Fig 2) . We selected one host protein, identified as tetraspan The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/331835 doi: bioRxiv preprint nsP1a interacts with CD63 in vivo. We transfected HEK293 T cells with 113 PEF-HA-nsP1a and pcDNA3.1-3flag-CD63.After 48 h, we performed 114 immunoprecipitation with the cell lysates from those cells using an anti-Flag M2 115 Affinity Gel (Sigma, St. Louis, MO, USA). We analyzed the resulting protein 116 complexes by western blot with anti-Flag or anti-HA antibody (Fig. 3A) . We 117 detected no proteins in control lysates from cells transfected with empty vector 118 (Fig. 3A , lane 1).We detected CD63 protein only in the lysates from cells that 119 wre transfected with pcDNA3.1-3flag-CD63 (Fig. 3A, lane 2) .Likewise, we 120 detectednsP1a protein only in the lysates from cells that were transfected with 121 PEF-HA-nsP1a (Fig. 3A, lane 3) . Both nsP1a and CD63 were readily detected in 122 the lysates from cellsthat were co-transfected with pcDNA3.1-3flag-CD63 and 123 PEF-HA-nsP1a. The HA antibody was able to pull down CD63-Flag together 124 with nsP1a-HA (Fig. 3A, lane4) . Likewise, the Flag antibody was able to pull 125 down nsP1a-HA together with CD63-Flag (Fig. 3A, lane4) . Taken together, the 126 results suggested that nsP1a was able to interact with the CD63 in vivo. 127 nsP1a interacts with CD63 in vitro. To further investigate the interaction 128 between nsP1a and CD63, we determined whether the interaction occurs 129 invitro.We expressed recombinant full-length nsP1a-GST fusion protein 130 (≈118kDa) induced by isopropyl β-D-thiogalactopyranoside(IPTG)in 131 Escherichia coli (Fig. 3B lanes 1-6) . We also detected purified nsP1afrom the 132 bacteria (Fig. 3B lane7) and human CD63 His Tag commercialized recombinant 133 protein (≈26kDa) by western blot (Fig